1. Field of the Invention
The present invention relates to an antigen including a polypeptide of an extracellular loop of the multi-transmembrane protein, and a method for screening and preparing the antibodies therewith.
2. Description of the Related Art
Since transmembrane proteins exist in human genome in large numbers (more than 7,000 kinds) and are importantly involved in disease-related phenomena such as cell signaling, they have been major target antigens for antibody therapy. As proteins that pass through the cell membrane 1 or 2 times, there are receptor proteins (about 400 kinds), enzyme proteins (about 500 kinds) and other cell membrane proteins (about 500 kinds). Also, there are various multi-transmembrane proteins that pass through the cell membrane 3 or more times, including receptor proteins (about 1000 kinds), most transport proteins (about 800 kinds) and proteins necessary for tight junction between cells. In order to develop useful antibodies using various transmembrane proteins, it is essential to secure antigens of high quality that maintain the natural state. In particular, for protein antigens, it is known that high-quality antibodies can be prepared when not only purity of protein but also 3-dimensional structure and physiological activity thereof are ensured. Although many researchers have prepared antigens using various transmembrane proteins, it was difficult to obtain superior antibodies for transmembrane proteins since they are mostly linear. Since antibodies are prepared mostly for the N-terminal or C-terminal region exposed to the cytoplasm or out of the cell membrane, they do not exert sufficient effect desired for the antibodies. Although transmembrane proteins are a very good target for research of diseases since they play important roles between cells or tissues, proteins prepared, for example, by recombination often have two or more transmembrane domains and do not maintain the natural state of being bound to the membrane in vivo. Further, coexistence of lipid components makes it difficult to obtain high-quality antibody. Many researchers have merely used expression products of transmembrane proteins obtained through a biological process or those obtained through selective synthesis of specific regions as antigens. Other researchers are studying fusion of transmembrane proteins with Fc of human antibodies for expression using animal cells in order to prepare antigens of various transmembrane proteins. However, the probability of the expressed proteins being expressed well in the animal cells and being presented as transmembrane proteins is low and a lot of time and cost are required for the development. Since multi-transmembrane proteins having two or more transmembrane domains naturally have an extracellular loop, the inventors of the present disclosure have devised a method of mimicking the regions and preparing a single-loop peptide using, for example, a linker for use as an antigen.
An antibody is a protein produced by the immune system. With high specificity for antigens as compared to small molecules, antibodies inactivate or remove antigens and thus can be a very efficient drug with few side effects. For example, transmembrane proteins exhibiting specific expression profile on the surface of cancer cells may be used as a target for antibody therapy for killing the cancer cells using immune cells or Immunomodulators. Also, the antibody may be used to selectively treat only the cancer cells as a carrier of radioisotopes or toxins (antibody-directed enzyme prodrug therapy; ADEPT). Although human antibodies are ideal for the antibody therapy, development thereof has been retarded due to the inability of production thereof by the hybridoma technology using human cells. Recently, the phage display technique that enables the production of human monoclonal antibodies without resorting to the human hybridoma technology is gaining a lot of attentions.
Researches are being focused on the claudin family with regard to the relationship between transmembrane proteins and cancer. Claudins are a family of cell membrane proteins with a molecular weight of approximately 20-34 kDa which have four transmembrane domains and constitute tight junctions. The claudin family includes 23-24 members in human and mice and each member of the claudins is known to exhibit a very unique expression pattern depending on each epithelial cell type (Furuse and Tsukita, Trends in Cell Biology 16: 181 (2006); Wilcox, et al., Cell 104: 165 (2001); Rahner, et al., Gastroenterology 120: 411 (2001)). In the sheet of epithelial cells, a mechanism works to prevent substances from leaking (diffusing) in the intercellular spaces and cell-cell adhesion systems called the tight junctions have been shown to actually play a critical role as a “barrier” in the mechanism to prevent leakage. A general structure of the claudin protein is described in detail in FIG. 1 (Lai-Nag M et. al Genomic Biology 2009, 10: 235 (2009)). It is reported that the expression of the claudin protein is closely related with cancer. In particular, it is known that claudin-1 suppresses breast cancer and prostate cancer and decreased expression of claudin-7 in esophageal carcinoma leads to increase of cancer cells owing to loss of E-cadherin. Since increased or decreased expression of the highly specific claudin protein in human cancer tissues is indicative of carcinogenesis, it may be a useful biomarker for detection, diagnosis and treatment of cancer. In particular, the expression of CLDN 3 and 4 is increased in various cancers and it is actively studied as a biomarker for ovarian cancer for which no useful drug has been developed yet (Choi et al., Histol Histopathol 22: 1185-1195 (2007)). Most antibodies developed using CLDN 3 and 4 are based on linear imaginary peptides as antigens.
CD151, also known as PETA-3 or SFA-1, is a protein in the tetraspanin family (Boucheix and Rubinstein, Cell Mol. Life Sci. 58: 1189-1205 (2001); Korean Patent Application Publication No. 2011-0010708; Hemler M E, J. Cell Biol. 155: 1103-1107 (2001)). In human, CD151 has 253 amino acids and includes four membrane fragments and two extracellular domains EC1 (18 amino acids, sequences 40-57) and EC2 (109 amino acids, sequences 113-221) which are called extracellular loops. In the nucleotide sequence of CD151, two variants of CD151 have been identified hitherto, i.e. one having nucleotides A and C at positions 395 and 409, respectively (Fitter et al., Blood 86 (4):1348-1355 (1995)) and the other having nucleotides G and T instead of the nucleotides A and C (Hasegawa et al., J. Virol. 70 (5): 3258-3263 (1996)). They have been identified as mutation of the residues K (Lys) and P (Pro) to the residues R (Arg) and S (Ser) at the positions 132 and 137 in the peptide sequence, respectively. CD151 interacts on the cell surface with various membrane proteins. Specifically, highly stable complexes resistant to the action of certain detergents with laminin receptor integrins, more specifically with integrin α3β1 or α6β4, whose preferred ligand is laminin 5, have been identified (Yauch et al., Mol. Biol. Cell 9:2751-2765 (1998); Lammerding et al., Proc. Natl. Acad. Sci. USA 100:7616-7621 (2003)). The association of these complexes involves the extracellular domains of CD151 and integrin. The QRD sequence [194-196] of CD151, located in the ECL2 loop, is very important in that association because mutation of this site causes loss of interaction with certain integrins (Kazarov et al., J. Cell Biol. 158: 1299-1309 (2002)).
Some previous researches have shown that overexpression of CD151 is associated with aggressiveness of certain cancers, such as lung, colon and prostate cancer, and that it might be considered to be a factor for poor prognosis (Tokuhara et al., Clin. Cancer Res. 7: 4109-4114 (2001); Hashida et al., Br. J. Cancer 89: 158-167 (2003); Ang et al., Cancer Epidemiol. Biomarkers Prev. 13: 1717-1721 (2004)). In these cases, mean survival rate was in fact decreased in those patients having tumors which express CD151, compared to those having tumors which do not express CD151. The overexpression of CD151 in various human tumor lines (HeLa, RPMI14788, A172, HT1080), brought about by transfection of the corresponding gene, causes increase in motility, migration and invasion of the transfected cells. Since these phenomena are inhibited in the presence of anti-CD151 antibodies (Testa et al., Cancer Res. 59: 3812-3820 (1999); Kohno et al., Int. J. Cancer 97: 336-343 (2002)), they can be a very important target in the development of anti-cancer drugs.
CD9 is also a cell surface glycoprotein receptor belonging to the tetraspanin family with a molecular weight of about 24-27 kD and is known to regulate signal transduction events playing important roles in development, activity and motility of cells. In addition, CD9 is known to be capable of regulating cell adhesion (Anton, E. S., et al., J. Neurosci. 15: 584-595, 1995) and cell migration (Klein-Soyer, C., et al., Arterioscler Thromb Vasc Biol. 20: 360-369, 2000) and triggering platelet activation and aggregation which are involved in platelet-induced endothelial cell proliferation (Masellis Smith, A., and Shaw, A. R., J. Immunol. 152: 2768-2777 (1994)). Moreover, it is known to be involved in various cellular phenomena such as promotion of muscle cell fusion and myotube maintenance. It is reported that the second extracellular loop (extracellular loop 2; ECL2) of CD9 is important in cell adhesion (George, A., et al., Blood 100: 4502-4511, 2002) and in promoting activity of DTR toward diphtheria toxin (DT) (Hidetoshi, H., et al. 289: 782-790 (2001)). In addition, although the ECL2 domain is glycosylated in many other tetraspanin family proteins, CD9 is distinguished in that the ECL1 domain is glycosylated. CD9 is reported to be related with cell motility and tumor metastasis (Miyake, M. and Hakomori, S., Biochemistry 30: 3328-3334, (1991)). It is presumed that CD9 exhibits tissue-specific aspects in cancer. Decreased expression of CD9 was observed in colon cancer (Mori, M., et al., Clin. Cancer Res. 4: 1507-1510 (1998)), breast cancer (Miyake, M., et al., Cancer Res. 55: 4127-4131 (1995)), lung cancer (Higachiyama, M., et al., Cancer Res. 55: 6040-6044 (1995); Funakoshi, T., et al., Oncogene 22: 674-687 (2003)) and pancreatic cancer (Sho, M., et al., Int. J. Cancer 79: 509-516 (1998)) patients and it is reported that it is associated with invasion, metastasis and poor prognosis of patients. However, there are some reports that the expression of CD9 is increased in head and neck squamous cell carcinoma (Erovic, B. M., et al., Head Neck 25: 848-857 (2003)) and stomach cancer (Hori, H., et al., J. Surg. Res. 117: 208-215 (2004)) with the progression of cancer.
Uroplakin 1B (UPK1B) is another member of the human tetraspanin family. It is a cell surface protein characterized by the presence of four hydrophobic domains. The protein mediates signal transduction events that play a role in the regulation of cell development, activation, growth and motility. In particular, interaction between the asymmetrical unit membrane (AUM) and the cytoskeleton is related with cancer and the relationship between the methylation of a CpG island with the UPK1B promoter and bladder cancer is well known (Varga A E, Leonardos L, Jackson P, Marreiros A, Cowled P A., Neoplasia. March-April; 6 (2): 128-35 (2004)).
Recently, the above-described membrane proteins are reported to be related with cancers. Although antibodies are being developed using specific regions thereof, they are mostly prepared based on linear imaginary peptides or partially expressed proteins as antigens.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.